CN116640742A - Acid phosphatase mutant, application thereof and method for preparing nicotinamide ribose by using acid phosphatase mutant - Google Patents
Acid phosphatase mutant, application thereof and method for preparing nicotinamide ribose by using acid phosphatase mutant Download PDFInfo
- Publication number
- CN116640742A CN116640742A CN202310310048.1A CN202310310048A CN116640742A CN 116640742 A CN116640742 A CN 116640742A CN 202310310048 A CN202310310048 A CN 202310310048A CN 116640742 A CN116640742 A CN 116640742A
- Authority
- CN
- China
- Prior art keywords
- acid phosphatase
- mutant
- seq
- parent
- enzyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010051457 Acid Phosphatase Proteins 0.000 title claims abstract description 117
- 102000013563 Acid Phosphatase Human genes 0.000 title claims abstract description 115
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 title claims abstract description 111
- 238000000034 method Methods 0.000 title claims abstract description 66
- JLEBZPBDRKPWTD-TURQNECASA-O N-ribosylnicotinamide Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](CO)O2)O)=C1 JLEBZPBDRKPWTD-TURQNECASA-O 0.000 title claims abstract description 64
- 239000011618 nicotinamide riboside Substances 0.000 claims abstract description 59
- 102000004190 Enzymes Human genes 0.000 claims abstract description 58
- 108090000790 Enzymes Proteins 0.000 claims abstract description 58
- 235000020956 nicotinamide riboside Nutrition 0.000 claims abstract description 58
- 238000006243 chemical reaction Methods 0.000 claims abstract description 40
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 29
- 238000002360 preparation method Methods 0.000 claims abstract description 29
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 29
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 21
- 239000000758 substrate Substances 0.000 claims abstract description 18
- 230000003197 catalytic effect Effects 0.000 claims abstract description 10
- DAYLJWODMCOQEW-TURQNECASA-O NMN(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(O)=O)O2)O)=C1 DAYLJWODMCOQEW-TURQNECASA-O 0.000 claims abstract 6
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 65
- 239000000243 solution Substances 0.000 claims description 63
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 39
- 229910052759 nickel Inorganic materials 0.000 claims description 32
- 230000008569 process Effects 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 239000000047 product Substances 0.000 claims description 21
- 239000000706 filtrate Substances 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 238000001914 filtration Methods 0.000 claims description 15
- 108010093096 Immobilized Enzymes Proteins 0.000 claims description 14
- 238000006911 enzymatic reaction Methods 0.000 claims description 14
- 239000013612 plasmid Substances 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 13
- 238000004108 freeze drying Methods 0.000 claims description 10
- LUEWUZLMQUOBSB-FSKGGBMCSA-N (2s,3s,4s,5s,6r)-2-[(2r,3s,4r,5r,6s)-6-[(2r,3s,4r,5s,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5s,6r)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](OC3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-FSKGGBMCSA-N 0.000 claims description 9
- 229920002581 Glucomannan Polymers 0.000 claims description 9
- 229920002752 Konjac Polymers 0.000 claims description 9
- 239000003480 eluent Substances 0.000 claims description 9
- 229940046240 glucomannan Drugs 0.000 claims description 9
- 235000010485 konjac Nutrition 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 230000035772 mutation Effects 0.000 claims description 8
- 239000011347 resin Substances 0.000 claims description 8
- 229920005989 resin Polymers 0.000 claims description 8
- 239000007790 solid phase Substances 0.000 claims description 8
- 238000005516 engineering process Methods 0.000 claims description 7
- 238000007852 inverse PCR Methods 0.000 claims description 7
- 238000011068 loading method Methods 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 6
- 238000001976 enzyme digestion Methods 0.000 claims description 6
- 239000000945 filler Substances 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 238000001471 micro-filtration Methods 0.000 claims description 6
- 239000001512 FEMA 4601 Substances 0.000 claims description 5
- HELXLJCILKEWJH-SEAGSNCFSA-N Rebaudioside A Natural products O=C(O[C@H]1[C@@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1)[C@@]1(C)[C@@H]2[C@](C)([C@H]3[C@@]4(CC(=C)[C@@](O[C@H]5[C@H](O[C@H]6[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O6)[C@@H](O[C@H]6[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O6)[C@H](O)[C@@H](CO)O5)(C4)CC3)CC2)CCC1 HELXLJCILKEWJH-SEAGSNCFSA-N 0.000 claims description 5
- 239000011543 agarose gel Substances 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- HELXLJCILKEWJH-UHFFFAOYSA-N entered according to Sigma 01432 Natural products C1CC2C3(C)CCCC(C)(C(=O)OC4C(C(O)C(O)C(CO)O4)O)C3CCC2(C2)CC(=C)C21OC(C1OC2C(C(O)C(O)C(CO)O2)O)OC(CO)C(O)C1OC1OC(CO)C(O)C(O)C1O HELXLJCILKEWJH-UHFFFAOYSA-N 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- HELXLJCILKEWJH-NCGAPWICSA-N rebaudioside A Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HELXLJCILKEWJH-NCGAPWICSA-N 0.000 claims description 5
- 235000019203 rebaudioside A Nutrition 0.000 claims description 5
- 238000012163 sequencing technique Methods 0.000 claims description 5
- 229910021586 Nickel(II) chloride Inorganic materials 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 4
- 239000007791 liquid phase Substances 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- QMMRZOWCJAIUJA-UHFFFAOYSA-L nickel dichloride Chemical compound Cl[Ni]Cl QMMRZOWCJAIUJA-UHFFFAOYSA-L 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 238000012795 verification Methods 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 3
- 239000012620 biological material Substances 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 239000012071 phase Substances 0.000 claims description 3
- 239000004793 Polystyrene Substances 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000010979 pH adjustment Methods 0.000 claims description 2
- 229920002223 polystyrene Polymers 0.000 claims description 2
- 239000013598 vector Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 15
- DAYLJWODMCOQEW-TURQNECASA-N NMN zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)([O-])=O)O2)O)=C1 DAYLJWODMCOQEW-TURQNECASA-N 0.000 description 23
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 10
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 239000012528 membrane Substances 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 6
- 238000001728 nano-filtration Methods 0.000 description 5
- 244000247812 Amorphophallus rivieri Species 0.000 description 4
- 235000001206 Amorphophallus rivieri Nutrition 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 4
- 239000000252 konjac Substances 0.000 description 4
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- DFPAKSUCGFBDDF-UHFFFAOYSA-N nicotinic acid amide Natural products NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 3
- 239000012064 sodium phosphate buffer Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 229920002873 Polyethylenimine Polymers 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- -1 more preferably Substances 0.000 description 2
- 229950006238 nadide Drugs 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000000053 physical method Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000011085 pressure filtration Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000000859 sublimation Methods 0.000 description 2
- 230000008022 sublimation Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- YABIFCKURFRPPO-IVOJBTPCSA-N 1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyridin-1-ium-3-carboxamide;chloride Chemical compound [Cl-].NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](CO)O2)O)=C1 YABIFCKURFRPPO-IVOJBTPCSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- PUEDDPCUCPRQNY-ZYUZMQFOSA-N D-ribosylnicotinate Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1[N+]1=CC=CC(C([O-])=O)=C1 PUEDDPCUCPRQNY-ZYUZMQFOSA-N 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 102100035703 Prostatic acid phosphatase Human genes 0.000 description 1
- 229930003537 Vitamin B3 Natural products 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 229940101270 nicotinamide adenine dinucleotide (nad) Drugs 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000001223 reverse osmosis Methods 0.000 description 1
- 150000008223 ribosides Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 235000019160 vitamin B3 Nutrition 0.000 description 1
- 239000011708 vitamin B3 Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03002—Acid phosphatase (3.1.3.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/385—Pyrimidine nucleosides
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention relates to the technical fields of molecular biology and bioengineering, and provides an acid phosphatase mutant and application thereof, and a method for preparing nicotinamide ribose, and aims to solve the technical problem that the cost of industrially producing nicotinamide ribose by an enzyme method is high due to low activity of wild acid phosphatase, and the acid phosphatase mutant is provided as protein in the following (a), (b) or (c): (a) the amino acid sequence is shown in SEQ ID NO: 3; (b) at the amino acid sequence set forth in SEQ ID NO:3, and has specific amino acid sequence shown in SEQ ID NO:2, a protein derived from (a) having a high catalytic activity as the acid phosphatase parent; the preparation method of nicotinamide riboside provided by the invention can ensure that the conversion rate of substrate nicotinamide mononucleotide can reach 99%, and the purity of prepared nicotinamide riboside can reach more than 99%.
Description
Technical Field
The invention relates to the technical field of molecular biology and bioengineering, in particular to an acid phosphatase mutant artificially obtained by a gene site-directed mutagenesis method, application thereof and a method for preparing nicotinamide riboside.
Background
Acid phosphatase (acid phosphatase) is a type of hydrolase that catalyzes the hydrolysis of a phosphomonoester (nucleotide, protein, etc.) molecule to remove a phosphate group, dephosphorylates a target molecule, and is most effective in an acidic environment, thus obtaining the name acid phosphatase, and EC number is EC 3.1.3.2.
Nicotinamide riboside (nicotinamide riboside, NR for short), also known as nicotinamide riboside, nicotinic acid riboside/glycoside, nicotinamide riboside, beta-D-nicotinamide riboside, CAS number 1341-23-7, has the structural formula shown below.
Nicotinamide riboside is a precursor to Nicotinamide Adenine Dinucleotide (NAD) and represents a source of vitamin B3. Studies have shown that supplementation with nicotinamide riboside can increase intracellular NAD concentration, thereby serving to prevent and ameliorate various unhealthy conditions resulting from NAD deficiency.
However, nicotinamide riboside contains high energy glycosidic linkages, which are spontaneously unstable in aqueous environments and decompose into nicotinamide and riboside products. This spontaneous decomposition can occur over a period of hours or days depending on the specific environmental conditions, making it difficult to maintain any naturally occurring nicotinamide riboside in the food, and to isolate nicotinamide riboside from natural sources, and thus it is currently common to produce nicotinamide riboside by chemical synthesis at home and abroad.
US 2017/011746 A1 discloses a method for producing nicotinamide riboside, which takes nicotinamide mononucleotide and/or derivatives thereof, water and a specific solvent as raw materials to produce nicotinamide riboside under the catalysis of phosphoric monoester hydrolase. The method adopts an enzyme method to prepare the nicotinamide riboside, has the advantage of simpler environment-friendly and healthier process than a chemical synthesis method, and is a development trend of nicotinamide riboside production. However, the activity of the wild acid phosphatase is relatively low, so that the cost for producing nicotinamide riboside by an enzymatic method is high, and the improvement of the enzyme activity of the acid phosphatase is of great significance for promoting the enzymatic industrial production of nicotinamide riboside.
Disclosure of Invention
The invention aims to solve the technical problem that the cost of industrially producing nicotinamide riboside by an enzyme method is high due to low activity of wild acid phosphatase, and develop an acid phosphatase with improved activity and a method for preparing nicotinamide riboside by using the acid phosphatase.
To achieve the above object, the present invention provides an acid phosphatase mutant which is a protein of the following (a), (b) or (c):
(a) The amino acid sequence is shown in SEQ ID NO: 3;
(b) In the sequence set forth in SEQ ID NO:3, and has specific amino acid sequence shown in SEQ ID NO:2, a protein derived from (a) having a high catalytic activity as the acid phosphatase parent;
(c) The amino acid sequence of the protein defined in (a) or (b) has more than 90 percent of homology, and the nicotinamide mononucleotide is taken as a substrate and has a specific amino acid sequence shown in SEQ ID NO:2, and a protein having a high catalytic activity.
The acid phosphatase parent is derived from saccharomyces cerevisiae (Saccharomyces cerevisiae), and the gene sequence of the acid phosphatase parent is shown in SEQ ID NO:1, the amino acid sequence is shown as SEQ ID NO: 2.
Preferably, with the sequence set forth in SEQ ID NO:2, the mutant of the invention has at least one mutation in at least one of the following positions compared to the amino acid sequence of the acid phosphatase parent: bit 44, bit 115, bit 206 and bit 240.
More preferably, with the sequence set forth in SEQ ID NO:2, the mutant of the invention has at least one of the following mutations compared to the amino acid sequence of the acid phosphatase parent: K44C, K115F, K I, D I and S240E.
The invention also provides a preparation method of nicotinamide riboside, which comprises the following steps: in KCl and anhydrous MgCl 2 Catalyzing the conversion of nicotinamide mononucleotide into nicotinamide riboside by using acid phosphatase in the presence, wherein the acid phosphatase has an amino acid sequence shown in SEQ ID NO:2 or an acid phosphatase mutant of (a), (b) or (c) as follows:
(a) The amino acid sequence is shown in SEQ ID NO: 3;
(b) In the sequence set forth in SEQ ID NO:3, and has specific amino acid sequence shown in SEQ ID NO:2, a protein derived from (a) having a high catalytic activity as the acid phosphatase parent;
(c) The amino acid sequence of the protein defined in (a) or (b) has more than 90 percent of homology, and the nicotinamide mononucleotide is taken as a substrate and has a specific amino acid sequence shown in SEQ ID NO:2, and a protein having a high catalytic activity.
The conversion route of the preparation method of nicotinamide riboside of the invention is shown in the following formula.
Preferably, the preparation method of nicotinamide riboside of the invention comprises the following steps in sequence: 1) KCl and anhydrous MgCl 2 Dissolving in water to obtain KCl and MgCl 2 Is a mixed solution of (a) and (b); 2) Cooling to room temperature; 3) To KCl and MgCl 2 Adding nicotinamide mononucleotide into the mixed solution of (1) to dissolve and adjusting the pH value to 6-7; 4) Adding acid phosphatase and adjusting pH to 6.8-7.3; 5) The reaction is carried out at 37.+ -. 5 ℃ for at least 1.5 hours.
In order to prevent degradation of the substrate nicotinamide mononucleotide, more preferably, konjac glucomannan and/or rebaudioside a is added to the reaction system before the enzymatic reaction of converting nicotinamide mononucleotide into nicotinamide riboside by the acid phosphatase is started. More preferably, konjac glucomannan is added.
More preferably, konjac glucomannan and/or rebaudioside a is added in an amount of 0.1 to 0.5 times the weight of nicotinamide mononucleotide.
The method of the invention firstly prepares KCl and anhydrous MgCl 2 The substrate nicotinamide mononucleotide is added after being dissolved and cooled to room temperature, which has the advantages that: the substrate is prevented from being affected by the excessively high heat generated during the dissolution process, thereby preventing the reduction of the conversion rate.
Preferably, the water in step 1) of the method of the invention is double distilled water, so as to reduce the influence of impurities in the water on the substrate and reduce the conversion rate. Double distilled water is obtained by distilling once distilled water again, and is called double distilled water, abbreviated as ddwater, and also abbreviated as ddH 2 O。
Preferably, naOH is used to adjust the pH in step 3) and step 4) of the process of the invention.
Preferably, the final concentration of nicotinamide mononucleotide in the reaction system in the method of the invention is 100+/-50 g/L.
Preferably, the final concentration of KCl in the reaction system in the process of the present invention is 15.+ -.5 g/L.
Preferably, mgCl in the process of the invention 2 The final concentration in the reaction system was 40.+ -.10 g/L.
In the method, the acid phosphatase parent is derived from saccharomyces cerevisiae (Saccharomyces cerevisiae), and the gene sequence of the acid phosphatase parent is shown in SEQ ID NO:1, the amino acid sequence is shown as SEQ ID NO: 2.
The acid phosphatase used in the method of the present invention may be commercially available enzymes purchased from the market, or may be self-prepared enzyme products, wherein the enzyme products may be crude enzyme forms without purification, or may be enzymes subjected to complete or partial purification, and the acid phosphatase of the present invention may be self-prepared by the following methods:
preparation of acid phosphatase parent: the PCR technology is used for the nucleotide sequence shown in SEQ ID NO:1, amplifying the gene sequence shown in the formula 1; then inserting the amplified product into NdeI and EcoRI sites of an expression vector pET22b (+) to obtain a recombinant plasmid pET22b-NS3; after sequencing verification, the recombinant plasmid pET22b-NS3 is transformed into escherichia coli, and is cultured and induced to express; and after the expression is finished, collecting thalli for cell disruption treatment, and collecting supernatant fluid to obtain crude enzyme liquid of the acid phosphatase parent.
Preparation of acid phosphatase mutant: designing a primer sequence of a mutation site by using an inverse PCR technology; inverse PCR amplification is carried out by using the designed primer and the recombinant plasmid pET22b-NS3 as a template; then the amplified product is treated by a Dpn I enzyme digestion template and then is transformed into escherichia coli; culturing and inducing the transformed escherichia coli; and after the expression is finished, collecting thalli for cell disruption treatment, and collecting supernatant fluid to obtain crude enzyme liquid of the acid phosphatase mutant.
Preferably, the acid phosphatase used in the method of the present invention is a purified enzyme solution obtained by purifying a crude enzyme solution of the acid phosphatase parent and/or mutant.
Preferably, the crude enzyme solution of the acid phosphatase parent and/or mutant is subjected to a purification treatment by a nickel column.
Preferably, the method for purifying the crude enzyme solution of the acid phosphatase parent and/or mutant by using a nickel column comprises the following steps: 1) Preparing a nickel column: using agarose gel as nickel carrier, using nickel chloride solution to hang nickel, using 1 XPBS+10 mM imidazole solution to balance nickel column; 2) Loading: uniformly mixing the crude enzyme solution and nickel filler, and then incubating in an ice bath for 1-2h; then loading the incubated mixture into the nickel column prepared in the step 1); 3) Eluting: first eluting with 1 XPBS+10 mM imidazole solution; and then eluting for the second time by using 1 XPBS+ (200-500) mM imidazole solution, and collecting eluent to obtain pure enzyme solution.
Of course, the above-described method of purifying with a nickel column is equally applicable to purifying crude enzyme solutions of other acid phosphatases than the acid phosphatase parent and mutant of the present invention.
In the above method for purifying crude enzyme solution, the first elution is performed for removing the impurity protein, and the second elution is performed for eluting the acid phosphatase protein.
Preferably, the acid phosphatase in the method of the present invention is an immobilized enzyme. The immobilized enzyme is an enzyme that has a catalytic action within a certain space and can be repeatedly and continuously used. In general, the enzyme-catalyzed reaction is carried out in an aqueous solution, and the immobilized enzyme is obtained by treating a water-soluble enzyme physically or chemically so as to be insoluble in water, but still having an enzymatic activity. The general stability of the immobilized enzyme is increased, the immobilized enzyme is easy to separate from a reaction system, is easy to control, can be repeatedly used, is convenient to transport and store, and is favorable for automatic production.
The immobilized enzyme used in the method of the invention can be purchased directly from the market, can be prepared by itself, and can be immobilized by itself after being purchased. Various enzyme immobilization carriers known in the art can be selected for immobilization, such as traditional inorganic carrier materials, such as silicon dioxide, activated carbon, glass beads and the like, and organic polymer carriers, such as macroporous poly N-aminoethylacrylamide-polyethylene and the like. In the present invention, the acid phosphatase is preferably immobilized using a nickel material or a solid-phase resin.
Preferably, the method for immobilizing acid phosphatase with the solid phase resin comprises: adding 0.2-0.8. 0.8M K to acid phosphatase 2 HPO 4 Adjusting pH to 7.5-9.5, adding solid phase resin, stirring at room temperature for 12-24 hr, and filtering to remove filtrate to obtain the immobilized enzyme of acid phosphatase.
Of course, the above-described immobilization method is equally applicable to immobilization of other acid phosphatases than the acid phosphatase parent and mutant of the present invention.
Preferably, the preparation method of nicotinamide riboside provided by the invention further comprises a post-treatment process of purifying an enzyme reaction solution for catalyzing the conversion of nicotinamide mononucleotide into nicotinamide riboside by acid phosphatase to obtain a refined nicotinamide riboside product, and the post-treatment process comprises the following steps: 1) Filtering the enzyme reaction solution, and collecting filtrate; 2) The pH value of the filtrate is regulated to be 5.0 plus or minus 0.5 so as to improve the stability of nicotinamide riboside in the solution; 3) Carrying out microfiltration on the filtrate subjected to the pH adjustment in the step 2), and collecting the filtrate; 4) Purifying the micro filtrate obtained in the step 3) by using a preparation liquid phase, wherein the preparation liquid phase uses polystyrene as a filler of a preparation column, uses ethanol and water as mobile phases, performs gradient elution, and collects eluent of a target peak; 5) And (3) freeze-drying the eluent collected in the step (4) to obtain the nicotinamide riboside refined product.
The filtration in the method of the invention refers to a process of separating solid and liquid in solution by adopting a physical method, and common filtration methods are applicable to the invention, including normal pressure filtration, reduced pressure filtration, centrifugal filtration and the like. Preferably, in the step 1) of the above post-treatment process, the enzyme reaction solution is filtered using a 200-400 mesh filtration membrane to remove the acid phosphatase from the enzyme reaction solution.
Microfiltration is also called microporous filtration, which uses microporous filter membrane as filter medium, under the pressure of 0.1-0.3 MPa, particles and bacteria between 0.1-1 micrometer are trapped, but macromolecular organic matter and inorganic salt can be allowed to pass through. Preferably, the filtrate is microfiltered in step 3) of the above post-treatment process using a microfiltration membrane of 0.45 μm.
Preferably, in step 4) of the above post-treatment process, the flow rate of the mobile phase is 40ml/min, the detection wavelength is 260nm, and the gradient of elution is: 0-13min, 0% ethanol and 100% water; 13-23min, ethanol 1% and water 99%;23-31min, ethanol 5% and water 95%;31-43min, 20% of ethanol and 80% of water; 43-45min, 20% of ethanol and 80% of water; 45-60min, ethanol 100% and water 0%.
Preferably, before the freeze-drying in step 5) of the above post-treatment process, the pH of the collected eluate is adjusted to 3-4 with hydrochloric acid, at which pH all nicotinamide riboside reacts with hydrochloric acid to form nicotinamide riboside chloride.
In order to obtain nicotinamide riboside with higher stability and better properties, preferably, the collected eluate is added with konjak glucomannan and/or rebaudioside a and mixed homogeneously before the freeze-drying in step 5) of the above post-treatment process.
More preferably, the konjac glucomannan and/or rebaudioside a is added in an amount of 1 to 2 times, even more preferably 1.5 times the weight of nicotinamide riboside.
In order to increase the efficiency of the freeze-drying and reduce the time of the freeze-drying, the collected eluate is preferably subjected to a concentration treatment to remove a large amount of water before the freeze-drying in step 5) of the above-mentioned post-treatment process. The concentration in the present invention means a process of increasing the concentration of a solution by evaporating a solvent by a physical method, and includes a reduced pressure distillation method, an ultrafiltration method, a dialysis method, an adsorption method, a freeze-drying method, and the like. Preferably, the eluate is concentrated to a concentration of nicotinamide riboside of 40-90g/L.
In order to prevent degradation of nicotinamide riboside, the concentration treatment process is preferably controlled to be carried out at a temperature below 25 ℃; more preferably, the concentration process is controlled to be performed at a temperature of 20 ℃ or less.
More preferably, the concentration treatment described above employs nanofiltration concentration.
Nanofiltration is a pressure-driven membrane separation process between reverse osmosis and ultrafiltration, a nanofiltration membrane is used as a filter medium, the pore size range of the nanofiltration membrane is about a few nanometers, and solvent molecules or solutes or low-valence ions with smaller relative molecular mass are allowed to permeate, so that the effects of separation and concentration are achieved.
Preferably, the sublimation temperature in the freeze-drying process of step 5) of the above post-treatment process is controlled below 25 ℃. Preferably, the moisture content in the nicotinamide riboside finished product is below 2% by freeze drying.
The invention also provides a method for preparing the acid phosphatase immobilized enzyme, which comprises the following steps:
1) Preparation of an acid phosphatase parent comprising: amplifying the gene sequence of the acid phosphatase parent by a PCR technology; then inserting the amplified product into an expression vector to obtain a recombinant plasmid; after sequencing verification, the recombinant plasmid is transformed into a host cell and is cultured and induced to express; collecting cells after the expression is finished, performing cell disruption treatment, and collecting supernatant fluid to obtain crude enzyme liquid of the acid phosphatase parent;
2) Preparation of acid phosphatase mutant, comprising: designing a primer sequence of a mutation site by using an inverse PCR technology; performing inverse PCR amplification by using the designed primer and the recombinant plasmid in the step 1) as a template; then the amplified product is treated by Dpn I enzyme digestion template and then is transformed into host cells, and is cultivated and induced to express; collecting cells after the expression is finished, and collecting supernatant fluid after the cell disruption treatment to obtain crude enzyme liquid of the acid phosphatase mutant;
3) Purifying the crude enzyme solution of the acid phosphatase parent and/or mutant by using a nickel column, wherein the method comprises the following steps: a) Preparing a nickel column: using agarose gel as nickel carrier, using nickel chloride solution to hang nickel, using 1 XPBS+10 mM imidazole solution to balance nickel column; b) Loading: uniformly mixing crude enzyme liquid of acid phosphatase parent and/or mutant with nickel filler, and incubating in ice bath for 1-2h; then loading the incubated mixture into the nickel column prepared in the step a); c) Eluting: first eluting with 1 XPBS+10 mM imidazole solution; then eluting for the second time by using 1 XPBS+ (200-500) mM imidazole solution, and collecting eluent to obtain pure enzyme solution of acid phosphatase parent and/or mutant;
4) Immobilization with a solid phase resin, comprising: adding 0.2-0 to the purified enzyme solution obtained in the step 3).8M K 2 HPO 4 The pH value is regulated to 7.5-9.5, then solid phase resin is added, stirring is carried out for 12-24 hours at room temperature, and the acid phosphatase immobilized enzyme is obtained after filtering and removing filtrate.
In the above-described method for producing an acid phosphatase immobilized enzyme, the acid phosphatase is the acid phosphatase parent or mutant of the present invention, and may be other acid phosphatase parent or mutant than the acid phosphatase parent or mutant of the present invention.
The invention also provides a biological material comprising a recombinant vector, a recombinant cell or a recombinant microorganism, which contains a gene encoding the acid phosphatase mutant according to the invention.
The invention also provides application of the acid phosphatase mutant in preparation of nicotinamide riboside.
The beneficial effects are that:
compared with the acid phosphatase parent, the acid phosphatase mutant provided by the invention has obviously improved enzyme activity, temperature stability and pH stability. The preparation method of nicotinamide riboside provided by the invention can ensure that the conversion rate of substrate nicotinamide mononucleotide can reach 99%, and the purity of prepared nicotinamide riboside can reach more than 99%.
Drawings
FIG. 1 is a diagram showing the construction process of recombinant plasmid pET22b-NS3 in example 1 of the present invention;
FIG. 2 is an HPLC chart of an enzyme reaction solution of mutant K115I of example 7;
FIG. 3 is an HPLC plot of the parent enzyme reaction solution of example 8;
FIG. 4 is an HPLC chart of an enzyme reaction solution of mutant K115I of example 9.
Detailed Description
The present invention will be described in further detail with reference to the following examples and the accompanying drawings, which are illustrative of the present invention, and the present invention is not limited to the following examples, which are given under conventional conditions or manufacturer's recommended conditions without specifying the specific conditions. Unless otherwise indicated, all materials and other chemicals used in the examples of the present invention are commercially available.
Example 1
Preparation of the acid phosphatase parent.
Saccharomyces cerevisiae (Saccharomyces cerevisiae) was derived from the primers 5 '-GGAATTCCATATGATGACCATTGCGAAGGATTACCGT-3' and 5 '-CCGGAATTCTTAGTGGTGGTGGTGGTGGT-3' as set forth in SEQ ID NO:1 (the amino acid sequence of which is shown as SEQ ID NO: 2) is amplified by a PCR amplification technology, and the obtained PCR amplification product is subjected to enzyme digestion treatment and is simultaneously inserted into NdeI and EcoRI sites of an expression vector pET22b (+) to obtain a recombinant plasmid pET22b-NS3, which is shown as the attached figure 1. After sequencing verification, the recombinant plasmid was transferred into E.coli Rosetta (de 3). The obtained recombinant escherichia coli is inoculated in a small-volume LB culture medium (containing 100 mug/mL of Amp), cultured overnight at 30-37 ℃, transferred into a certain volume LB culture medium (containing 100 mug/mL of Amp) with an inoculum size of 1-5%, continuously cultured at 30-37 ℃ until the OD600 reaches 0.6-1.0, added with isopropyl-beta-D-thiogalactoside (IPTG) with a final concentration of 0.1-1 mM, induced to express at 20-37 ℃ for 10-20 h, and centrifugally collected.
Example 2
Preparation of acid phosphatase mutant
The following mutation sites were designed: K44C, K115F, K115I, D206I, S240E, K I/S240E, K F/K44C/D206I, K I/S240E/D206I, inverse PCR was performed using the mutant primer sequences as shown in Table 1, and the recombinant plasmid pET22b-NS3 constructed in example 1 as a template, and the PCR system and PCR procedure were as follows:
PCR system:
PCR procedure:
after the PCR product is purified by a gel recovery kit, the enzyme digestion is carried out by using Dpn I, the enzyme digestion product is transferred into escherichia coli DH5 alpha, and after screening by an Amp plate, bacterial colony is picked up for sequencing, and the mutant recombinant plasmid which is successfully mutated at the site of K44C, K115F, K115I, D206I, S240E, K I/S240E, K F/K44C/D206I, K115I/S240E/D206I is confirmed to be obtained and transferred into escherichia coli Rosetta (de 3). Inoculating the obtained mutant recombinant escherichia coli into a small-volume LB culture medium (containing 100 mug/mL of Amp), culturing overnight at 30-37 ℃, transferring the obtained product into a certain-volume LB culture medium (containing 100 mug/mL of Amp) with an inoculum size of 1-5%, continuously culturing at 30-37 ℃ until the OD600 reaches 0.6-1.0, adding isopropyl-beta-D-thiogalactoside (IPTG) with a final concentration of 0.1-1 mM, and centrifuging to collect thalli after induction expression at 20-37 ℃ for 10-20 hours.
TABLE 1
Example 3
Preparation of crude enzyme solution of acid phosphatase
Re-suspending the bacteria collected by fermentation by using a1 XPBS+10 mM imidazole solution with the mass of 4 times, and then crushing the bacteria by using a high-pressure homogenizer; after homogenization is completed, adding Polyethylenimine (PEI) with the total volume of 0.5-1% under stirring and adjusting the pH to 7.0-8.0; the supernatant clarified enzyme solution was collected by centrifugation, and the enzyme solution was filtered with a 0.45. Mu.L filter membrane, and the filtrate was collected to obtain a crude enzyme solution of acid phosphatase.
Example 4
Nickel column preparation
a) After the column was cleaned, agarose gel was used as nickel carrier according to nickel packing (ml): agarose gel was prepared at a ratio of cell (g) =1:2, and ddH was used 2 O and 1 XPBS were washed sequentially with 5 column volumes each for agarose gelGlue;
b) Preparing a nickel chloride solution with the concentration of 0.2mol/L, and then hanging nickel, wherein in order to fully hang nickel, repeated hanging can be carried out;
c) ddH with 5 column volumes 2 O washes off unbound Ni 2+ The nickel column was equilibrated with 5-10 column volumes of 1 XPBS+10 mM imidazole solution (pH=8.0).
Example 5
Loading and elution
a) The crude enzyme solution of the acid phosphatase obtained in the example 3 is evenly mixed with nickel filler and then transferred to a conical flask, and incubated for 1-2h in a shaking table ice bath at the rotating speed of 100rm;
b) Eluting the hybrid protein with 1 XPBS+10 mM imidazole solution (pH=8.0), then fully eluting the target protein with 1 XPBS+200 mM imidazole solution (pH=8.0), and collecting the eluent to obtain the pure enzyme solution of the acid phosphatase.
Example 6
Enzyme activity comparison experiment
The method for measuring the enzyme activity comprises the following steps: KCl at a final concentration of 200mM and MgCl at 400mM 2 As cofactor, NMN with a final concentration of 2mmol is added into a 200uL reaction system, then 10uL diluted pure enzyme solution is added, a blank control replaces the pure enzyme solution with a corresponding buffer solution, after uniformly mixing and reacting for 10min at 37 ℃ and pH7.0, 200uL of 10% trichloroacetic acid solution is immediately added for uniformly mixing, the reaction is stopped, the sample is diluted by 50 times by methanol, and the amount of the product is detected by HPLC, and the enzyme activity unit U is calculated.
Definition of acid phosphatase enzyme Activity U: the amount of enzyme required to hydrolyze substrate NMN to release 1umol in 1 minute at 37℃and pH7.0 is referred to as an International Unit of enzyme Activity (U).
The comparison experiment results of the enzyme activities and the stability of the acid phosphatase parent and the acid phosphatase mutant are shown in Table 2, wherein the temperature stability and the pH stability refer to the preservation range that the residual activity is more than or equal to 90% after the target enzyme is stored for 12 hours under the corresponding preservation conditions.
TABLE 2
The results show that compared with the acid phosphatase parent, the acid phosphatase mutant has obviously improved enzyme activity, temperature stability and pH stability.
Example 7
Preparation of nicotinamide riboside
Designing a total system of 2L, setting the final concentration of a substrate to be 100g/L, preparing a clean 3L three-mouth bottle, and adding 1L ddH 2 O, then 30g KCl and 80g anhydrous MgCl are added 2 Stirring to dissolve, cooling to room temperature, adding 200g NMN, and adjusting pH to 6-7 with 5M NaOH to dissolve completely. The reaction temperature was set at 37℃and 900ml of the pure enzyme solution of the acid phosphatase mutant K115I obtained in example 5 was added, and the 2L system was supplemented with sodium phosphate buffer at pH7.0, and the reaction system was adjusted to pH7.2 to start the reaction. After 2 hours of reaction, the reaction solution is diluted by pure water for 50-100 times, the reaction result is analyzed by high performance liquid chromatography after microporous filtration, the analysis method is shown in table 3, and the HPLC chart is shown in figure 2. The detection result shows that: the degradation of the substrate NMN is more pronounced, about 42% of the substrate is degraded, and the conversion of the remaining NMN to NR is 99.9%.
TABLE 3 Table 3
Example 8
Designing a total system of 2L, setting the final concentration of a substrate to be 100g/L, preparing a clean 3L three-mouth bottle, and adding 1L ddH 2 O, then 30g KCl and 80g anhydrous MgCl are added 2 Stirring to dissolve, cooling to room temperature, adding 20g of konjak glucomannan, adding 200g of NMN, and adjusting pH to 6-7 with 5M NaOH to dissolve completely. The reaction temperature was set at 37℃and 900ml of the pure enzyme solution of the acid phosphatase mutant K115I obtained in example 5 was added, and the 2L system was supplemented with sodium phosphate buffer at pH7.0, and the reaction system was adjusted to pH7.2 to start the reaction. After 2 hours of reaction, the reaction solution is diluted by pure water for 50 to 100 times, and after microporous filtration, the reaction result is analyzed by high performance liquid chromatography and separatedThe analytical method is shown in Table 3, and the HPLC chart is shown in FIG. 3. The detection result shows that: the substrate NMN is not degraded obviously, and the conversion rate of NMN to NR is 99.8%.
Example 9
Designing a total system of 2L, setting the final concentration of a substrate to be 100g/L, preparing a clean 3L three-mouth bottle, and adding 1L ddH 2 O, then 30g KCl and 80g anhydrous MgCl are added 2 Stirring to dissolve, cooling to room temperature, adding 20g of konjak glucomannan, adding 200g of NMN, and adjusting pH to 6-7 with 5M NaOH to dissolve completely. The reaction temperature was set at 37℃and 900ml of the pure enzyme solution of the acid phosphatase parent obtained in example 5 was added, and the reaction was started by adding 2L of the pure enzyme solution to the reaction system with sodium phosphate buffer at pH7.0 and adjusting the reaction system to pH 7.2. After 2 hours of reaction, the reaction solution is diluted by pure water for 50-100 times, the reaction result is analyzed by high performance liquid chromatography after microporous filtration, the analysis method is shown in table 3, and the HPLC chart is shown in figure 4. The detection result shows that: the substrate NMN is not significantly degraded, and the conversion rate of NMN to NR is 72.5%.
Example 10
After the catalytic reaction of nicotinamide riboside is completed, filtering enzyme reaction liquid through 200-400 mesh filter cloth to remove acid phosphatase therein, adding 2-6M hydrochloric acid into the obtained filtrate to adjust the PH to about 5.0, carrying out microfiltration through a microporous filter membrane with the thickness of 0.45 mu M, and collecting the microfiltration liquid; purifying the micro filtrate with the preparation solution, wherein the control conditions of the preparation solution are shown in table 4, and collecting the eluent of NR section to obtain the aqueous solution with nicotinamide riboside purity of more than 99%; then carrying out nanofiltration concentration on the obtained nicotinamide riboside aqueous solution with the purity of more than 99 percent at the temperature of below 25 ℃ to increase the concentration of nicotinamide riboside to 50-60g/L, and then adding hydrochloric acid to adjust the PH to 3-4; adding konjak glucomannan into a nicotinamide ribose aqueous solution with the concentration of 90g/L, fully dissolving and stirring uniformly; drying in a freeze drier, controlling sublimation temperature below 25deg.C, and obtaining white powdery nicotinamide riboside refined product with purity above 99% after 24 hr. The yields and purities of the nicotinamide riboside refined products obtained by separating and purifying the enzyme reaction solutions of examples 7, 8, and 9 are shown in Table 5.
TABLE 4 Table 4
TABLE 5
Claims (12)
1. An acid phosphatase mutant, characterized in that: the mutant is a protein of the following (a), (b) or (c):
(a) The amino acid sequence is shown in SEQ ID NO: 3;
(b) In the sequence set forth in SEQ ID NO:3, and has specific amino acid sequence shown in SEQ ID NO:2, a protein derived from (a) having a high catalytic activity as the acid phosphatase parent;
(c) The amino acid sequence of the protein defined in (a) or (b) has more than 90 percent of homology, and the nicotinamide mononucleotide is taken as a substrate and has a specific amino acid sequence shown in SEQ ID NO:2, and a protein having a high catalytic activity.
2. The acid phosphatase mutant according to claim 1, wherein the amino acid sequence of SEQ ID NO:2, said mutant having at least one mutation in at least one of the following positions: bit 44, bit 115, bit 206 and bit 240.
3. The acid phosphatase mutant according to claim 1, wherein the amino acid sequence of SEQ ID NO:2, said mutant having at least one of the following mutations compared to the amino acid sequence of the acid phosphatase parent shown in seq id no: K44C, K115F, K I, D I and S240E.
4. A first partA process for the preparation of nicotinamide riboside, characterized in that it comprises: in KCl and anhydrous MgCl 2 In the presence of acid phosphatase, which is an amino acid sequence shown in SEQ ID NO:2 or an acid phosphatase mutant which is a protein of the following (a), (b) or (c):
(a) The amino acid sequence is shown in SEQ ID NO: 3;
(b) In the sequence set forth in SEQ ID NO:3, and has specific amino acid sequence shown in SEQ ID NO:2, a protein derived from (a) having a high catalytic activity as the acid phosphatase parent;
(c) The amino acid sequence of the protein defined in (a) or (b) has more than 90 percent of homology, and the nicotinamide mononucleotide is taken as a substrate and has a specific amino acid sequence shown in SEQ ID NO:2, and a protein having a high catalytic activity.
5. The method for preparing nicotinamide riboside according to claim 4, characterized in that it comprises the following steps in order: 1) KCl and anhydrous MgCl 2 Dissolving in water to obtain KCl and MgCl 2 Is a mixed solution of (a) and (b); 2) Cooling to room temperature; 3) To KCl and MgCl 2 Adding nicotinamide mononucleotide into the mixed solution of (1) to dissolve and adjusting the pH value to 6-7; 4) Adding acid phosphatase and adjusting pH to 6.8-7.3; 5) The reaction is carried out at 37.+ -. 5 ℃ for at least 1.5 hours.
6. A process for the preparation of nicotinamide riboside according to claim 4 or 5, characterized in that it further comprises: before the acid phosphatase catalyzes the enzymatic reaction of converting nicotinamide mononucleotide into nicotinamide riboside, konjak glucomannan and/or rebaudioside A is added into the reaction system.
7. The method for preparing nicotinamide riboside according to claim 4 or 5, wherein: the acid phosphatase is added in the form of pure enzyme liquid, and the pure enzyme liquid is a solution containing the acid phosphatase, which is obtained by performing induced expression on a microorganism containing the acid phosphatase gene, performing cell disruption and centrifugation to remove precipitated crude enzyme liquid, and performing nickel column purification treatment.
8. The method for producing nicotinamide riboside according to claim 4 or 5, wherein the acid phosphatase is an immobilized enzyme, and the immobilized enzyme is produced by: adding 0.2-0.8. 0.8M K to the acid phosphatase 2 HPO 4 Adjusting pH to 7.5-9.5, adding solid phase resin, stirring at room temperature for 12-24 hr, and filtering to remove filtrate.
9. The method for preparing nicotinamide riboside according to claim 4 or 5, wherein: the method also comprises a post-treatment process for purifying from an enzyme reaction solution of acid phosphatase for catalyzing the conversion of nicotinamide mononucleotide into nicotinamide riboside to obtain a refined nicotinamide riboside product, wherein the post-treatment process comprises the following steps of: 1) Filtering the enzyme reaction solution, and collecting filtrate; 2) Regulating the pH value of the filtrate to be 5.0+/-0.5; 3) Carrying out microfiltration on the filtrate subjected to the pH adjustment in the step 2), and collecting the filtrate; 4) Purifying the micro filtrate obtained in the step 3) by using a preparation liquid phase, wherein the preparation liquid phase uses polystyrene as a filler of a preparation column, uses ethanol and water as mobile phases, performs gradient elution, and collects eluent of a target peak; 5) And (3) freeze-drying the eluent collected in the step (4) to obtain the nicotinamide riboside refined product.
10. A method of preparing an acid phosphatase immobilized enzyme comprising the acid phosphatase mutant according to claim 1, 2 or 3 and a parent thereof, the method comprising:
1) Preparation of an acid phosphatase parent comprising: amplifying the gene sequence of the acid phosphatase parent by a PCR technology; then inserting the amplified product into an expression vector to obtain a recombinant plasmid; after sequencing verification, the recombinant plasmid is transformed into a host cell and is cultured and induced to express; collecting cells after the expression is finished, performing cell disruption treatment, and collecting supernatant fluid to obtain crude enzyme liquid of the acid phosphatase parent;
2) Preparation of acid phosphatase mutant, comprising: designing a primer sequence of a mutation site by using an inverse PCR technology; performing inverse PCR amplification by using the designed primer and the recombinant plasmid in the step 1) as a template; then the amplified product is treated by Dpn I enzyme digestion template and then is transformed into host cells, and is cultivated and induced to express; collecting cells after the expression is finished, and collecting supernatant fluid after the cell disruption treatment to obtain crude enzyme liquid of the acid phosphatase mutant;
3) Purifying the crude enzyme solution of the acid phosphatase parent and/or mutant by using a nickel column, wherein the method comprises the following steps: a) Preparing a nickel column: using agarose gel as nickel carrier, using nickel chloride solution to hang nickel, using 1 XPBS+10 mM imidazole solution to balance nickel column; b) Loading: uniformly mixing crude enzyme liquid of acid phosphatase parent and/or mutant with nickel filler, and incubating in ice bath for 1-2h; then loading the incubated mixture into the nickel column prepared in the step a); c) Eluting: first eluting with 1 XPBS+10 mM imidazole solution; then eluting for the second time by using 1 XPBS+ (200-500) mM imidazole solution, and collecting eluent to obtain pure enzyme solution of acid phosphatase parent and/or mutant;
4) Immobilization with a solid phase resin, comprising: adding 0.2-0.8. 0.8M K to the purified enzyme solution obtained in the step 3) 2 HPO 4 The pH value is regulated to 7.5-9.5, then solid phase resin is added, stirring is carried out for 12-24 hours at room temperature, and the acid phosphatase immobilized enzyme is obtained after filtering and removing filtrate.
11. A biological material comprising a recombinant vector, a recombinant cell or a recombinant microorganism, characterized in that: the biological material contains a gene encoding the acid phosphatase mutant according to claim 1, 2 or 3.
12. Use of the acid phosphatase mutant according to claim 1, 2 or 3 for the preparation of nicotinamide riboside.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310310048.1A CN116640742A (en) | 2018-08-17 | 2018-08-17 | Acid phosphatase mutant, application thereof and method for preparing nicotinamide ribose by using acid phosphatase mutant |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310310048.1A CN116640742A (en) | 2018-08-17 | 2018-08-17 | Acid phosphatase mutant, application thereof and method for preparing nicotinamide ribose by using acid phosphatase mutant |
| CN201880038009.8A CN110770339B (en) | 2018-08-17 | 2018-08-17 | Acid phosphatase mutant, application thereof and method for preparing nicotinamide ribose by using acid phosphatase mutant |
| PCT/CN2018/101174 WO2019210606A1 (en) | 2018-08-17 | 2018-08-17 | Acid phosphatase mutant, application thereof, and method for preparing nicotinamide riboside |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201880038009.8A Division CN110770339B (en) | 2018-08-17 | 2018-08-17 | Acid phosphatase mutant, application thereof and method for preparing nicotinamide ribose by using acid phosphatase mutant |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116640742A true CN116640742A (en) | 2023-08-25 |
Family
ID=68386996
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310310048.1A Pending CN116640742A (en) | 2018-08-17 | 2018-08-17 | Acid phosphatase mutant, application thereof and method for preparing nicotinamide ribose by using acid phosphatase mutant |
| CN201880038009.8A Active CN110770339B (en) | 2018-08-17 | 2018-08-17 | Acid phosphatase mutant, application thereof and method for preparing nicotinamide ribose by using acid phosphatase mutant |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201880038009.8A Active CN110770339B (en) | 2018-08-17 | 2018-08-17 | Acid phosphatase mutant, application thereof and method for preparing nicotinamide ribose by using acid phosphatase mutant |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US11299721B2 (en) |
| JP (1) | JP6893000B2 (en) |
| CN (2) | CN116640742A (en) |
| WO (1) | WO2019210606A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109097351A (en) * | 2018-08-01 | 2018-12-28 | 浙江海洋大学 | A kind of preparation method of the novel immobilised enzymes for oil spilling reparation |
| JP7213603B2 (en) * | 2020-06-19 | 2023-01-27 | ボンタック バイオエンジニアリング（シェンゼン） カンパニー リミテッド | Method for preparing nicotinamide mononucleotide using nicotinamide as raw material |
| CN115814857B (en) * | 2022-09-06 | 2024-03-26 | 华南理工大学 | Artificial metalloenzyme based on Nitrophorin2 protein scaffold and preparation method and application thereof |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2931999A1 (en) * | 1979-08-03 | 1981-02-26 | Schering Ag | PRODUCTION AND USE OF RECOMBINED PLASMIDES WITH GENES FOR ALKALINE PHOSPHATASES |
| JP4304727B2 (en) * | 1996-11-21 | 2009-07-29 | 味の素株式会社 | Method for producing nucleoside-5'-phosphate ester |
| CN100575487C (en) * | 2007-03-29 | 2009-12-30 | 复旦大学 | A kind of method of production 5 '-flavour nucleotide |
| BRPI0922980A8 (en) * | 2008-12-18 | 2017-06-20 | Versitech Ltd | METHOD TO ACCELERATE PLANT GROWTH AND IMPROVE PRODUCTION BY INTRODUCING PHOSPHATASE INTO TRANSGENIC PLANT |
| CN102382871A (en) * | 2011-03-10 | 2012-03-21 | 上海复昌科技有限公司 | Improved method for producing 5'-flavor nucleotide |
| CN102206617A (en) * | 2011-03-30 | 2011-10-05 | 清华大学 | Acidic phosphatase, its encoding gene and application |
| CN104342418B (en) * | 2013-07-24 | 2017-05-31 | 东莞泛亚太生物科技有限公司 | The phytase of the enzymatic activity with lifting |
| CN103555687B (en) * | 2013-09-12 | 2015-06-17 | 浙江工业大学 | Acid phosphatase mutant, encoding gene, vector and application |
| US20170121746A1 (en) | 2015-10-28 | 2017-05-04 | The Procter & Gamble Company | Method For Preparing Nicotinamide Riboside |
-
2018
- 2018-08-17 CN CN202310310048.1A patent/CN116640742A/en active Pending
- 2018-08-17 CN CN201880038009.8A patent/CN110770339B/en active Active
- 2018-08-17 WO PCT/CN2018/101174 patent/WO2019210606A1/en active Application Filing
- 2018-08-17 US US16/623,942 patent/US11299721B2/en active Active
- 2018-08-17 JP JP2019565518A patent/JP6893000B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| US11299721B2 (en) | 2022-04-12 |
| US20200224183A1 (en) | 2020-07-16 |
| JP2020521473A (en) | 2020-07-27 |
| WO2019210606A1 (en) | 2019-11-07 |
| CN110770339A (en) | 2020-02-07 |
| CN110770339B (en) | 2023-06-02 |
| JP6893000B2 (en) | 2021-06-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107267575B (en) | Method for producing N-acetyl-D-glucosamine and/or D-glucosamine salt by microbial fermentation | |
| JP5455244B2 (en) | Method for producing galactooligosaccharides by free cells | |
| CN110770339B (en) | Acid phosphatase mutant, application thereof and method for preparing nicotinamide ribose by using acid phosphatase mutant | |
| CN108866028B (en) | Amino lyase mutant protein, and coding gene and application thereof | |
| CN107208085B (en) | Immobilized cell and preparation method thereof | |
| CN113249372B (en) | Preparation method and application of immobilized cells for mannose production | |
| JP2009225705A (en) | Method for producing theanine | |
| CN118755681B (en) | Cytochrome P450 BM3 mutant and application thereof | |
| CN116254279B (en) | Method for catalyzing xylitol to biosynthesize L-xylose by using double-enzyme cascade recombinant escherichia coli | |
| CN117625585B (en) | Bifunctional enzyme NagEA and application thereof | |
| CN117778371B (en) | Co-immobilized enzyme of phenylpyruvate decarboxylase and alcohol dehydrogenase, preparation and application | |
| KR101252928B1 (en) | Immobilized enzyme using pH-sensitive polymer and preparation method of linear alpha-1,4-glucans using the same | |
| CN118755681A (en) | Cytochrome P450 BM3 mutant and application thereof | |
| Chibata et al. | Immobilized cells | |
| CN117535281A (en) | Method for orderly immobilizing multiple enzymes by amino microspheres, product and application thereof | |
| CN117417923A (en) | Nitrilase mutant and application thereof | |
| CN116042599A (en) | In-vitro multienzyme system for producing 5-hydroxytryptophan | |
| JP2005087149A (en) | Method for producing d-chiro-inositol | |
| CN117343887A (en) | Lactobacillus plantarum lactose permease deletion mutant strain DeltalacS1DeltalacS2 and application thereof | |
| JP2627755B2 (en) | Production of acylneulaminate cytidyltransferase | |
| CN118853516A (en) | Recombinant bacterium for producing nicotinamide adenine dinucleotide phosphate, construction method and application thereof | |
| CN113754785A (en) | Fusion protein, preparation method thereof and application thereof in preparation of fucosylation product | |
| CN118910023A (en) | Nitrilase mutant and application thereof in production of D-mandelic acid | |
| CN118056902A (en) | Mature polypeptide sequence for synthesizing HMOs and application thereof | |
| CN114934040A (en) | Method for synthesizing isoprene by catalyzing mevalonic acid in vitro with immobilized enzyme |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |